SETDB1 modulates the TGFβ response in Duchenne muscular dystrophy myotubes

Overactivation of the transforming growth factor-β (TGFβ) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to fibrosis and muscle dysfunction. Here, we investigated the role of SETDB1 (SET domain, bifurcated 1), a histone lysine methyltransferase involved in muscle differentiation. Our data show that, following TGFβ induction, SETDB1 accumulates in the nuclei of healthy myotubes while being already present in the nuclei of DMD myotubes where TGFβ signaling is constitutively activated. Transcriptomics revealed that depletion of SETDB1 in DMD myotubes leads to down-regulation of TGFβ target genes coding for secreted factors involved in extracellular matrix remodeling and inflammation. Consequently, SETDB1 silencing in DMD myotubes abrogates the deleterious effect of their secretome on myoblast differentiation by impairing myoblast pro-fibrotic response. Our findings indicate that SETDB1 potentiates the TGFβ–driven fibrotic response in DMD muscles, providing an additional axis for therapeutic intervention.

A. Scheme of microscopy analysis and nuclear/cytoplasmic signal quantification.Nuclear masks were setup from DAPI pictures and applied on SETDB1 pictures and total cell masks were selected on SETDB1 pictures.Cytoplasmic quantification was performed by subtracting nuclei mask from total cell masks.B. Quantification of phospho-SMAD3 nuclear fluorescence in proliferating myoblasts, myotubes treated or not with TGFb1.C. Protein levels of SETDB1 and phospho-SMAD3 in total extracts of myoblasts and myotubes treated or not with TGFb1.D. RT-qPCR of early myogenic (MYOD1, Myogenin) and TGFb-related genes (TGFB1, SERPINE1) in myoblasts and myotubes treated or not with TGFb1.E. RT-qPCR of early myogenic marker MYOD1 and TGFb-related genes (SERPINE1, TGFB1) in myotubes treated or not with TGFb1 and/or its inhibitor SB-431542.For all panels: Statistics were performed on ≥3 biological replicates (>100 nuclei for immunostaining quantification) and data are represented as average +/-SEM *p<0.05;**p<0.01;***p<0.001(unpaired Student's t test). C.

Healthy
Figure S1: SETDB1 localization depends on TGFb/SMAD pathway activation during muscle terminal differentiation

Figure S2 :
Figure S2: SETDB1 translocate into muscle cell nuclei in response to TGFb/SMAD pathway activation and show more persistent nuclear signal in DMD myotubes irrespectively of the type of DMD mutation A. RT-qPCR of TGFB1 shows a higher response of DMD myotubes to TGFb1 treatment as compared to WT cells.B. Immunostaining of SETDB1 (red) in healthy#2 and DMD point mutation myotubes.Nuclei were stained with DAPI (blue).Scale bar, 10 µM. C. SETDB1 and MyHC immunostaining in proliferating muscle cells and in myotubes treated or not with TGFb1 derived from iPSCs of healthy or DMD individuals.Nuclei were stained with DAPI (blue).Scale bar, 10 µM.Diagrams represent the percentage of SETDB1 signal intensity measure in nuclei and cytoplasm of the cells.D-F.DMD del ex45 differentiating myotubes (48 h differentiation) were transfected with control scrambled (siCTL) or SMAD3 siRNA (siSMAD3).2 days later, myotubes were treated with TGFb and after 1 day, myotubes were subjected to different assays, as follows: D. Immunostaining of SETDB1 (red), and nuclei staining with DAPI (blue).Scale bar, 10 µm.E. Quantification of SETDB1 (n=2) nuclear/cytoplasmic IF signal ratio.F. RT-qPCR of SMAD3 mRNA in control siRNA (siCTL) and siRNA against SMAD3 (siSMAD3) conditions to check the siRNA efficiency (representative of n=2).G.Western blot showing SETDB1 and pSMAD3 protein migration profiles in nuclear (N), peri-nuclear (PN) and cytoplasmic (C) fractions of healthy myotubes.RNA polymerase II and Lamin A/C were used as controls for nuclear fraction and beta-Actin as a control of cytoplasmic fraction.For all panels: Statistics were performed on ≥3 biological replicates (>100 nuclei for immunostaining quantification) and data are represented as average +/-SEM *p<0.05;**p<0.01;***p<0.001(unpaired Student's t test).

Figure S4 :
Figure S4: Healthy and DMD myotubes respond differently to TGFb/SMAD pathway activation but display some SETDB1 target gene signatures in common A. Principal component analysis of the filtered (see Materials & Methods) RNA-seq data, depicting a well grouping of all the samples for each of the 6 experimental conditions of the study.The variability captured by the first PC corresponds to the healthy vs. DMD conditions and the one of the second PC to the TGFβ treatment.B. Gene Ontology enrichment of DEGs from healthy myotubes basal versus TGFβ comparison.C. Gene Ontology enrichment of DEGs from DMD myotubes basal versus TGFβ comparison.D. Gene Set Enrichment Analysis (GSEA) of DEGs from healthy myotubes TGFβ +/-siSETDB1.E. Validation by RT-qPCR of genes coding for proteins involved in ECM-remodeling (ADAMTS8, MMP14), TGFb/BMP and Wnt pathway (MSTN, BMPR2, WNT5A) and unknown function but predicted as regulator of muscle differentiation (ANKRD33B).F. TapeStation profile of ATAC libraries from healthy or DMD myotubes prepared using the ATAC-seq kit from Diagenode (ref: C01080001).For all panels: Statistics were performed on ≥3 biological replicates and data are represented as average +/-SEM *p<0.05;**p<0.01;***p<0.001(unpaired Student's t test).

Figure S5 :
Figure S5: Catalytic-dead SETDB1 induced a strong differentiation phenotype even at low expression levels.A. Western blot (WB) on DMD myotube protein extracts after 72 hours of treatment with the indicated different concentrations of doxycycline, showing protein levels of SETDB1 (endogenous and exogenous), FLAG (exogenous SETDB1), and b-actin used as loading controls.B. Diagram of the experimental plan.C. WB on DMD myotube protein extracts at different time points post-treatment with 1000 ng/mL doxycycline.D. Validation by RT-qPCR of SETDB1 total, endogenous and exogenous mRNA after 72h of doxycycline treatment.E. Polyclonal DMD myoblasts differentiation observed by phase contrast light microscopy.Scale bar, 50 µM.F. Immunostaining of SETDB1 (green).Nuclei were stained with DAPI (blue).Scale bar, 10 μm.G. Quantification of nuclei/myotubes in the presence or absence of WT SEDTB1 and catalytic-dead mutant H1224K SETDB1.

Figure S6 :•
Figure S6: TGFb/SMAD pathway activation leads to fusion defects in muscle cells A. Diagram of the experimental design.B.Immunofluorescence of pSMAD3 (green) and MHC (red) in myoblasts differentiated in fresh or conditioned medium +/-TGFb1.Nuclei were stained with DAPI (blue).Scale bar, 10 µM. C. Myogenic and fusion index of the myoblasts differentiated in fresh or conditioned medium +/-TGFb1.D. Raw myogenic and fusion index in basal conditioned medium show difference in differentiation rate between healthy and DMD myotubes.E. Quantification of fusion index after 6 days of differentiation in conditioned medium produced by healthy or DMD myotubes +/-siSETDB1 +/-TGFb1.F. RT-qPCR of pro-fibrotic genes, MSTN and SERPINE1, and muscle-related genes MYOD1, Myogenin, MYH1 and MCK after 3 days of differentiation in conditioned medium produced by healthy or DMD myotubes +/-siSETDB1.For all panels: Statistics were performed on ≥3 biological replicates (>100 nuclei for immunostaining quantification) and data are represented as average +/-SEM *p<0.05;**p<0.01;***p<0.001(unpaired Student's t test).